Moticam 2300 camera

To assess whether PST® stimulation could affect hTSCs phenotype, cell morphology was examined with a phase-contrast microscope (Axiovert 40 CFL, Zeiss, equipped with a Moticam 2300 camera, Motic) after 0, 10, 24, and 48 h of PST® exposure. For cell viability analyses, hTSCs were subjected to PST® stimulation, as described before. PST and control cells were analyzed at each time point after harvesting with Trypsin-EDTA solution (Sigma-Aldrich) by counting with a Countess Cell Counter (Invitrogen, Life Technologies), according to the manufacturer’s procedure. Cell viability was determined by trypan blue dye exclusion assay. The number of viable cells in each sample was expressed as a percentage of the total untreated cells number at day 0. All assays were carried out in triplicates for each sample.

hTSCs were plated at a concentration of 3 × 10⁴ cells/cm² in normal growth medium, and then switched to the osteogenesis induction medium, which was constituted of DMEM-low glucose (Sigma-Aldrich), 10 % FBS (HyClone, Thermo-Fisher Scientific), 4 mM L-glutamine (Euroclone), 1 % antibiotic-antimycotic mixture (Euroclone), supplemented with 0.1 μM dexamethasone, 50 μg/ml L-ascorbic acid-2-phosphate, and 10 mM β-glycerophosphate (all reagents from Sigma-Aldrich) for 17 days. At day 17, Alizarin Red solution (Millipore) was used to detect calcium deposition in derived osteoblasts, according to the manufacturer’s instruction. All photomicrographs were acquired with an Axiovert 40 microscope (Zeiss) equipped with a Moticam 2300 camera (Motic). The osteogenic medium was changed every 2–3 days.

Wound-healing assay was performed as previously described [26 (link)]. hTSCs were grown to confluence in 6-well plates and were subjected to PST® stimulation or were kept outside the incubator for the same amount of time (controls). A sterile P200 pipet tip was used to create a scratch across the cell monolayer. Then, cultures were washed once with 1 ml of growth medium to remove the damaged and detached cells. After replacing the medium, hTSCs were allowed to grow for 48 h. At different time points, cell cultures were examined with a phase-contrast microscope (Axiovert 40 CFL, Zeiss, equipped with a Moticam 2300 camera, Motic) and images of the same scratch fields were acquired at time 0 and after 5, 20, 24, and 30 h from the scratch. The gap area between the cells was calculated in each acquired image using software ImageJ. The migration rate was based on the measure of the recovered wound area (experimental data expressed in percentage). All assays were carried out in triplicates for each sample.